Neurological drug screens

ABSTRACT

Compositions for identifying lead compounds for pharmacological agents useful in the diagnosis or treatment of neurological disease or injury include mixtures comprising an isolated netrin and an isolated natural netrin receptor such as &#34;Deleted in Colorectal Carcinoma&#34; (DCC) and neogenin. The general methods involve incubating a mixture of an isolated mammalian netrin, an isolated natural mammalian netrin receptor, and a candidate pharmacological agent, and determining if the presence of the agent modulates the binding of the netrin to the receptor.

The research carried out in the subject application was supported inpart by grants from the National Institutes of Health. The governmentmay have rights in any patent issuing on this application.

RELATED APPLICATION

This application is a division of Ser. No. 08/551,874 filed Oct. 16,1996, issued as U.S. Pat. No. 5,747,262 on May 5, 1998.

INTRODUCTION

1. Field

The field of the invention is neurological drug screening usingmammalian netrins and natural mammalian netrin receptors.

2. Background

The netrins are a family of proteins that are profound modulators ofgrowth of developing axons, functioning as attractants for some axonsand repellents of other axons. As such, the modulation of these effectsprovides an important therapeutic pathway for assisting the regenerationof axons in adult nervous system (e.g. following injury or trauma).

The ability to construct high-throughput pharmaceutical screens formodulators of netrin activity has been limited by the lack ofidentifiable mammalian netrin receptors. Identifying receptors on axonsthat mediate neural responsiveness to netrins will provide key targetsfor identifying lead pharmaceuticals for therapeutic intervention in thenervous system.

Relevant Literature

Ishii et al. (1992) Neuron 9, 873-881, describe unc-6, a nematode genewith sequence similarity to mammalian netrin genes. Leung-Hagenstein etal. (1992) Cell 71, 289-299, Hedgecock et al. (1990) Neuron 2, 61-85 andHamelin et al. (1993) Nature 364, 327-330, describe two other genes,unc-5 and unc-40 that are in the same genetic pathway as unc-6 and havesequence similarity to the vertebrate genes, "Deleted in ColorectalCarcinoma" (DCC) and neogenin (Fearon et al. (1990) Science 247, 49-56,Hendrick et al. (1994), and Vielmetter et al. (1994) JCB 127,2009-2020). Cho et al. (1995) Current Opinion in Genetics andDevelopment 5, 72-78, provide a recent review of DCC function. See also:Colamarino & Tessier-Lavigne (1995) Cell 81, 621-629; Kennedy et al.(1994) Cell 78, 425-435; and Serafini et al. (1994) Cell 71, 289-299.

SUMMARY OF THE INVENTION

The invention provides methods and compositions for identifying leadcompounds for pharmacological agents useful in the diagnosis ortreatment of neurological disease or injury. In particular, theinvention provides mixtures comprising an isolated netrin and anisolated natural netrin receptor capable of specifically binding saidnetrin. Exemplary netrin receptors-include DCC and neogenin. The generalmethods involve incubating a mixture comprising an isolated netrin, anisolated natural netrin receptor, and a candidate pharmacological agent,and determining if the presence of the agent modulates the binding ofthe netrin to the receptor. Specific agents provide lead compounds forpharmacological agents useful in the diagnosis or treatment ofneurological disease or injury.

DETAILED DESCRIPTION

The invention provides methods and compositions for identifying leadcompounds for pharmacological agents useful in the diagnosis ortreatment of mammalian, particularly human, neurological disease orinjury. The methods rely on monitoring the interaction of a mammalian,particularly human, netrin and a corresponding netrin receptor in thepresence and absence of a candidate agent. A wide variety of assays canbe used, including receptor activation assays and binding assays.Binding assays may monitor netrin binding to the extracellular domain ofa full-length receptor expressed on a cell, or in vitro protein-proteinbinding of a netrin to a C-terminal truncated receptor. Typically, suchin vitro screens involve the immobilization of one of the bindingpartners on a solid substrate.

Invariably, the assays involve a mixture comprising an isolated netrinand an isolated natural netrin receptor capable of specifically bindingsaid netrin. Exemplary netrin receptors used in the assays include DCCand neogenin. We have demonstrated that these mammalian gene productsfunction as natural mammalian, and in particular, human, netrinreceptors. The general methods comprise steps: forming a mixturecomprising an isolated netrin, an isolated natural netrin receptor, anda candidate pharmacological agent; incubating said mixture underconditions whereby, but for the presence of said candidatepharmacological agent, said netrin specifically binds said netrinreceptor at a first binding affinity; and detecting a second bindingaffinity of said netrin to said netrin receptor, wherein a differencebetween said first and second binding affinity indicates that saidcandidate pharmacological agent is a lead compound for a pharmacologicalagent useful in the diagnosis or treatment of neurological disease orinjury.

The following examplary assay is offered by way of illustration and notby way of limitation: Ligand Screening of Transfected COS cells.

I. Prepare the Ligand

Expression Construct: cDNA encoding the targeted netrin is tagged withthe Fc--portion of human IgG and subcloned into a 293 expression vector(pCEP4: In Vitrogen).

Transfection: 293 EBNA cells are transfected (CaPO₄ method) with thenetrin expression construct. After 24 h recovery, transfected cells areselected with G418 (geneticin, 250 ug/ml, Gibco) and hygromycin (200ug/ml). Once the selection process is complete, cells are maintained inDulbecco's Modified Eagles medium (DME)/10% FCS under selection.

Preparation of Conditioned Medium: Serum-containing media is replacedwith Optimem with glutamax-1 (Gibco) and 300 ng/ml heparin (Sigma), andthe cells are conditioned for 3 days. The media is collected and spun at3,000×g for 10 minutes. The supernatant is filtered (0.45 um) and storedwith 0.1% azide at 4° C. for no more than 2 weeks.

II. Prepare Truncated Receptor (Positive Control)

Expression Construct: cDNA encoding a corresponding netrin receptordeletion mutant comprising the extracellular domain (truncatedimmediately N-terminal to the transmembrane region) is subcloned into a293 expression vector (pCEP4: In Vitrogen).

Transfection: 293 EBNA cells are transfected (CaPO₄ method) with thereceptor mutant expression construct. After 24 h recovery, transfectedcells are selected with G418 (geneticin, 250 ug/ml, Gibco) andhygromycin (200 ug/ml). Once the selection process is complete, cellsare maintained in Dulbecco's Modified Eagles medium (DME)/10% FCS underselection.

Preparation of Conditioned Medium: Serum-containing media is replacedwith Optimem with glutamax-1 (Gibco) and 300 ng/ml heparin (Sigma), andthe cells are conditioned for 3 days. The media is collected and spun at3,000×g for 10 minutes. The supernatant is filtered (0.45 um) and storedwith 0.1% azide at 4° C. for no more than 2 weeks.

II. Transfect COS Cells

Seed COS cells (250,000) on 35 mm dishes in 2 ml DME/10% FCS. 18-24 hlater, dilute 1 ug of netrin receptor-encoding DNA (cDNA cloned intopMT21 expression vector) into 200 ul serum-free media and add 6 ul ofLipofectamine (Gibco). Incubate this solution at room temperature for15-45 min.

Wash the cells 2X with PBS. Add 800 ul serum-free media to the tubecontaining the lipid-DNA complexes. Overlay this solution onto thewashed cells.

Incubate for 6 h. Stop the reaction by adding 1 ml DMA/20% FCS. Refeedcells. Assay cells 12 hr later.

III. Ligand Binding Assay

Wash plates of transfected COS cells 1X with cold PBS (plus Ca/Mg)/1%goat serum. Add 1 ml conditioned media neat and incubate 90 min at roomtemp.

Wash plates 3X with PBS (plus Ca/Mg). On the 4th wash, add 1 ml 50%methanol to 1 ml PBS. Then add 1 ml methanol. Evacuate and add 1 mlmethanol.

Wash 1X with PBS. Wash 1X PBS/1% goat serum.

Add secondary antibody (1-to-2,000 anti-human Fc conjugated to alkalinephosphatase (Jackson Lab)) in PBS/1% goat serum. Incubate 30-40 min roomtemp.

Wash 3X with PBS. Wash 1X alkaline phosphatase buffer (100 mM Tris-Cl,pH 9.5, 100 mM NaCl, 5 mM MgCl). Prepare alkaline phosphatase reagents:4.5 ul/ml NBT and 3.5 ul/ml BCIP (Gibco) in alkaline phosphatase buffer.

Incubate 10-30 min, quench with 20 mM EDTA in PBS. Cells that have boundnetrin are visible by the presence of a dark purple reaction product.

In parallel incubations, positive controls are provided by titratingnetrin binding with serial dilutions of the mutant receptor conditionedmedium.

IV. Results: Binding of Netrin to Netrin Receptor

Cell expressing mammalian netrin were shown to bind netrin receptor. Noreactivity was observed with control COS cells or withreceptor-expressing COS cells in the presence of the secondary antibodybut in the absence of the netrin-Fc fusion. Binding was observed toreceptor-expression cells using a construct in which netrin is fuseddirectly to alkaline phosphatase, for which a secondary antibody is notrequired. Receptor deletion mutants titrate the netrin-receptor binding,serving as a positive control for inhibition assays.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. Although the foregoing invention has beendescribed in some detail by way of illustration and example for purposesof clarity of understanding, it will be readily apparent to those ofordinary skill in the art in light of the teachings of this inventionthat certain changes and modifications may be made thereto withoutdeparting from the spirit or scope of the appended claims.

What is claimed is:
 1. A mixture comprising an isolated mammalian netrinand an isolated mammalian netrin receptor selected from the groupconsisting of "Deleted in Colorectal Carcinoma" (DCC) and neogenin.
 2. Amixture according to claim 1, wherein said receptor is "Deleted inColorectal Carcinoma" (DCC).
 3. A mixture according to claim 1, whereinsaid receptor is neogenin.
 4. A mixture according to claim 1, whereinsaid netrin is a human netrin and said receptor is "Deleted inColorectal Carcinoma" (DCC).
 5. A mixture according to claim 1, whereinsaid netrin is a human netrin and said receptor is neogenin.
 6. Amixture according to claim 1, wherein said netrin is a human netrin.